1. Real Time Electrochemical Displacement Immunosensor for TCA detection María Viviana Duarte 2nd year PhD Student Project thesis course January 2004

2. 5 W and 1 H Real Time Electrochemical Displacement Immunosensor for TCA detection- MV Duarte What? Real Time Electrochemical Displacement Immunosensor for TCA detection - 2,4,6 trichloroanisole (TCA) detection using antiTCA-IgG Who? MV Duarte- 2nd year PhD student. Project thesis When? PhD studies Where? BBG group – Etseq Universitat Rovira I Virgili Why? TCA - as an indicator of contaminants responsible for cork taint How? Our Tools- Antibodies and Electrochemical immunosensor

3. Cork taint incidents Why- An introduction Real Time Electrochemical Displacement Immunosensor for TCA detection- MV Duarte 2,4,6-trichloroanisole (TCA) An indicator! Cost money to cork and wine industry

4. Antibodies How- Our Tools Real Time Electrochemical Displacement Immunosensor for TCA detection- MV Duarte Immunosensors Selective immunodetection of TCA Displacement methodology: reagentless and fast responding devices

5. How- The detection Real Time Electrochemical Displacement Immunosensor for TCA detection- MV Duarte Antibody antiTCA- IgG 150 k DA TCA 0,2 k DA

6. Real Time Electrochemical Displacement Immunosensor for TCA detection- MV Duarte

7. Graphite electrode substrate e - product HRP labelled TCA Amperometric signal t/seg TCA-HRP 1- TCA- HRP Adsorption Real Time Electrochemical Displacement Immunosensor for TCA detection- MV Duarte Materials and Methods- System 1

8. t/seg TCA-HRP Graphite electrode substrate e- product HRP labelle d TCA 1- TCA- HRP Adsorption 2- TCA (analyte) addition Displacement Substrate addition TCA (analyte) additions Real Time Electrochemical Displacement Immunosensor for TCA detection- MV Duarte

9. System 1- Results TCA injections started TCP or HaptenA Specific and non-specific Adsorption Adsorption Amperograms TCA- HRP on IgG electrode Displacement chronoamperogram Real Time Electrochemical Displacement Immunosensor for TCA detection- MV Duarte 1.Substrate 2.TCA-HRP 1- TCA- HRP Adsorption2- TCA addition Substrate

10. e - product Amperometric signal t/seg Antimouse- HRP 1- antimouse- HRP Adsorption Real Time Electrochemical Displacement Immunosensor for TCA detection- MV Duarte Materials and Methods- System 2 substrate TCA-BSA

11. 1- Antimouse- HRP Adsorption 2- TCA (analyte) addition Displacement Real Time Electrochemical Displacement Immunosensor for TCA detection- MV Duarte e - product substrate

12. System 2- Results Displacement experiments are the next step! BSA electrode BSA or TCA-BSA 5%- Co Pos 90%-PEG 5%; 30 min 37ºC anti TCA IgG; chornoamperometric signal of antimouse HRP TCA-BSA electrode Real Time Electrochemical Displacement Immunosensor for TCA detection- MV Duarte

13. 50 nm Incident light Reflected light Real Time Electrochemical Displacement Immunosensor for TCA detection- MV Duarte 50 nm channel

14. Immobilized antiTCA antibody Hapten A- HRP Immobilized antiTCA antibody Hapten 1- HRP + + kon: 5,22 E+2 M-1sec-1 koff: 7,14 E-4 sec-1 koff: 4,72 E-3 sec-1 K Association=kon/koff K A (M-1) 7,31 E+5 1,90 E+5 Real Time Electrochemical Displacement Immunosensor for TCA detection- MV Duarte kon: 8,97 E+2 M-1sec-1

15. SPR Characterization: The affinity of immobilized AntiTCA IgG Clon 007 with Hapten A- HRP is 3,5 times higher than with Hapten1-HRP Immuno-electrochemical sensor: It seems that BSA- TCA electrodes (system 2) - AntiTCA IgG–antimouse HRP adsorbed could be use to study further TCA displacements Conclusions With the results obtained at the moment: Real Time Electrochemical Displacement Immunosensor for TCA detection- MV Duarte

16. Immunoelectrochemical sensor Continue with electrochemichal System 2 and the Displacement experiments Use SPR information to improve System 1 Antibody characterization- SPR Continue with the characterization of different anti TCA-IgG (clon) and their interaction with TCA – HRP conjugations, BSA- TCA conjugations, TCA, etc Future work Real Time Electrochemical Displacement Immunosensor for TCA detection- MV Duarte

17. Thank you for your attention Real Time Electrochemical Displacement Immunosensor for TCA detection- MV Duarte

18. TCA Hapten A Hapten 1 Haptens Real Time Electrochemical Displacement Immunosensor for TCA detection- MV Duarte